N-linked glycans, specific oligosaccharide structures attached to asparagine residues of glycoproteins, can contribute significantly to the properties of the protein and, in turn, to the properties of the organism. Plant proteins can carry N-linked glycans but in marked contrast to mammals only few biological processes are known to which they contribute.
Biogenesis of N-linked glycans begins with the synthesis of a lipid linked oligosaccharide moiety (Glc3Man9GlcNAc2-) which is transferred en bloc to the nascent polypeptide chain in the endoplasmic reticulum (ER). Through a series of trimming reactions by exoglycosidases in the ER and cis-Golgi compartments, the so-called “high mannose” (Man9GlcNAc2 to Man5GlcNAc2) glycans are formed. Subsequently, the formation of complex type glycans starts with the transfer of the first GlcNAc onto Man5GlcNAc2 by GnTI and further trimming by mannosidase II (MannII) to form GlcNAcMan3GlcNAc2. Complex glycan biosynthesis continues while the glycoprotein is progressing through the secretory pathway with the transfer in the Golgi apparatus of the second GlcNAc residue by GnTII as well as other monosaccharide residues onto the GlcNAcMan3GlcNAc2 under the action of several other glycosyl transferases.
Plants and mammals differ with respect to the formation of complex glycans (see FIG. 1, which compares the glycosylation pathway of glycoproteins in plants and mammals). In plants, complex glycans are characterized by the presence of β(1,2)-xylose residues linked to the Man-3 and/or an α(1,3)-fucose residue linked to GlcNAc-1, instead of an α(1,6)-fucose residue linked to the GlcNAc-1. Genes encoding the corresponding xylosyl (XyIT) and fucosyl (FucT) transferases have been isolated [Strasser et al., “Molecular cloning and functional expression of beta1,2-xylosyltransferase cDNA from Arabidopsis thaliana,” FEBS Lett. 472:105 (2000); Leiter et al., “Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha 1,3-fucosyltransferase from mung beans,” J. Biol. Chem. 274:21830 (1999)]. Plants do not possess β(1,4)-galactosyltransferases nor α(2,6)sialyltransferases and consequently plant glycans lack the β(1,4)-galactose and terminal α(2,6)NeuAc residues often found on mammalian glycans.
The final glycan structures are not only determined by the mere presence of enzymes involved in their biosynthesis and transport but to a large extent by the specific sequence of the various enzymatic reactions. The latter is controlled by discrete sequestering and relative position of these enzymes throughout the ER and Golgi, which is mediated by the interaction of determinants of the transferase and specific characteristics of the sub-Golgi compartment for which the transferase is destined. A number of studies using hybrid molecules have identified that the transmembrane domains of several glycosyltransferases, including that of β(1,4)-galactosyltransferases, play a central role in their sub-Golgi sorting [Grabenhorst et al., J. Biol. Chem 274:36107 (1999); Colley, K., Glycobiology 7:1 (1997); Munro, S., Trends Cell Biol. 8:11 (1998); Gleeson, P. A., Histochem. Cell Biol. 109:517 (1998)].
Although plants and mammals have diverged a relatively long time ago, N-linked glycosylation seems at least partly conserved. This is evidenced by the similar though not identical glycan structures and by the observation that a mammalian GlcNAcTI gene complements a Arabidopsis mutant that is deficient in GlcNAcTI activity, and vice versa. The differences in glycan structures can have important consequences. For example, xylose and α(1,3)-fucose epitopes are known to be highly immunogenic and possibly allergenic in some circumstances, which may pose a problem when plants are used for the production of therapeutic glycoproteins. Moreover, blood serum of many allergy patients contains IgE directed against these epitopes but also 50% of non-allergic blood donors contains in their sera antibodies specific for core-xylose whereas 25% have antibodies for core-alpha 1,3-fucose (Bardor et al., 2002, in press, Glycobiology) (Advance Access published Dec. 17, 2002) which make these individuals at risk to treatments with recombinant proteins produced in plants containing fucose and/or xylose. In addition, this carbohydrate directed IgE in sera might cause false positive reaction in in vitro tests using plant extracts since there is evidence that these carbohydrate specific IgE's are not relevant for the allergenic reaction. In sum, a therapeutic failure with a glycoprotein produced in plants might be the result of accelerated clearance of the recombinant glycoprotein having xylose and/or fucose.
Accordingly, there is a need to better control glycosylation in plants, and particularly, glycosylation of glycoproteins intended for therapeutic use.